This notebook implements TIMER to analyze immune infiltrate composition within TCGA samples. TIMER has a web portal that could give us the results for each of the desired tumor types (Ovarian, Breast, Colon, and Lung), but we are going to manually re-run the analysis so that we can compare the expression of the three papers’ human IFN-viral signatures to the enrichment of different immune subsets in TCGA samples for the same data. On the other hand, I can download the Cox PH model output from https://cistrome.shinyapps.io/timer/
We will locate the TIMER script (we won’t source it, because doing so runs the script right away), format the data as the tutorial asks (tutorial is here: http://cistrome.org/TIMER/misc/readme.txt)
The preparation required
TIMER analaysis was all performed on the Cancer Genome Atlas (TCGA) samples. The gene expression data profiled by RNA-seq or Affymetrix arrays are accessible through GDAC firehose (https://gdac.broadinstitute.org). Please first download all the data matrices (preferrably RSEM, if not available, then RPKM) and transform them into XXXX.Rdata format (where XXXX is the cancer abbreviation, in lower case, same below), then deposit into a folder named "RNAseq". Clinical data for all cancers should be downloaded from TCGA public ftp: https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/tumor/XXXX/bcr/nationwidechildrens.org/bio/clin/. Replace "XXXX" with the desired cancer abbreviation and select the latest version of the clinical file ending with "patient_XXXX.txt". Please put all the clinical data files into a folder named "Clinical". TIMER requires tumor purity information for acurate estimation of immune-related genes. Purity is available in the Related data files. After downloading the zip file, please unzip the purity estimation files into a folder named "AGP". TIMER also uses reference immune cell expression files to estimate the immune infiltration levels. These files are available in the Related data files 1-3. Please download these files and put them into a folder named "immune_datasets". Please also download the Supplementar Table S5 from Rooney et al., 2015, Cell and convert it to a text file (mmc5.txt) and place it into a folder named "virus". # install.packages("sva")
library(sva)
Loading required package: mgcv
package 'mgcv' was built under R version 3.4.4Loading required package: nlme
package 'nlme' was built under R version 3.4.4
Attaching package: 'nlme'
The following object is masked from 'package:IRanges':
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This is mgcv 1.8-24. For overview type 'help("mgcv-package")'.
Attaching package: 'mgcv'
The following object is masked from 'package:futile.logger':
scat
Loading required package: genefilter
Attaching package: 'genefilter'
The following object is masked from 'package:MASS':
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Loading required package: BiocParallel# Please first download all the data matrices (preferrably RSEM, if not available, then RPKM) and transform them into XXXX.Rdata format (where XXXX is the cancer abbreviation, in lower case, same below), then deposit into a folder named "RNAseq".
# downloaded GDAC files; now to load them
setwd('/Users/vinayakvsv/big_pqe/analysis/timer/')
# Breast
brca_gdac <- read.table(file = "../../datasets/gdac_firehose/gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0/BRCA.rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.data.txt",header = TRUE,sep = "\t",row.names = 1)
save(brca_gdac, file = "./data/RNAseq/brcaRNAseq.RData")
# Colon
coad_gdac <- read.table(file = "../../datasets/gdac_firehose/gdac.broadinstitute.org_COAD.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0/COAD.rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.data.txt",header = TRUE,sep = "\t",row.names = 1)
save(coad_gdac, file = "./data/RNAseq/coadRNAseq.RData")
# Lung
lusc_gdac <- read.table(file = "../../datasets/gdac_firehose/gdac.broadinstitute.org_LUSC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0/LUSC.rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.data.txt",header = TRUE,sep = "\t",row.names = 1)
save(lusc_gdac, file = "./data/RNAseq/luscRNAseq.RData")
# Ovarian
ov_gdac <- read.table(file = "../../datasets/gdac_firehose/gdac.broadinstitute.org_OV.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0/OV.rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.data.txt",header = TRUE,sep = "\t",row.names = 1)
save(ov_gdac, file = "./data/RNAseq/ovRNAseq.RData")
# save cancer files
gdac_cancer_files <- list(brca = brca_gdac,
coad = coad_gdac,
lusc = lusc_gdac,
ov = ov_gdac)# Clinical data for all cancers should be downloaded from TCGA public ftp: https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/tumor/XXXX/bcr/nationwidechildrens.org/bio/clin/. Replace "XXXX" with the desired cancer abbreviation and select the latest version of the clinical file ending with "patient_XXXX.txt". Please put all the clinical data files into a folder named "Clinical".
# update: get the clinical data from https://portal.gdc.cancer.gov/. Perhaps from GDAC firehose? YES, from firehose. Go here: http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/OV/20160128/
# we will use files like OV.clin.merged.txt and rename them "patient_XXXX.txt"
# done
# TIMER requires tumor purity information for acurate estimation of immune-related genes. Purity is available in the Related data files. After downloading the zip file, please unzip the purity estimation files into a folder named "AGP".
# related files are http://cistrome.org/TIMER/download.html
# TIMER also uses reference immune cell expression files to estimate the immune infiltration levels. These files are available in the Related data files 1-3. Please download these files and put them into a folder named "immune_datasets".
# Please also download the Supplementar Table S5 from Rooney et al., 2015, Cell and convert it to a text file (mmc5.txt) and place it into a folder named "virus".
# to convert the mmc5 file...
read_excel_allsheets <- function(filename, tibble = FALSE) {
# from https://stackoverflow.com/questions/12945687/read-all-worksheets-in-an-excel-workbook-into-an-r-list-with-data-frames
sheets <- excel_sheets(filename)
x <- lapply(sheets, function(X) readxl::read_excel(filename, sheet = X))
if(!tibble) x <- lapply(x, as.data.frame)
names(x) <- sheets
x
}
mmc5_rooney <- read_excel_allsheets(filename = "./virus/mmc5.xlsx")
# we likely want the first sheet
write.table(x = mmc5_rooney$S5A_CTantigenStats,file = "./virus/mmc5.txt")# 3. Data depository structure.
# Please put all the folders in step 1 into one folder named "data". Download the R codes and put both .R files at the same level of folder 'data'. Create a new folder at this level, named 'results/'. Under results/, create 23 folders, each with the name of the cancer abbreviation in lowercase. The analysis results for each cancer will be written in the corresponding folder. Please download Related data file 5 and put on the same level as the R codes. Upon finishing this step, your current folder should have 2 folders (data and results), 1 Rdata file and 2 R codes.
# Doneredblue <- read.table(file = "redblue100.txt",header = TRUE,sep = "\t",row.names = 1)
save(redblue, file = "./redblue.Rdata")# 4. Running TIMER.
# If you are interested in one cancer, please open an R console, type:
# cc='XXXX' ## replace XXXX with your desired cancer abbreviation
# cur.dir='Path_to_your_current_directory'
# source('CancerImmunePipeline.R')
#
# 5. Running TIMER in batch mode
# Open R console, type:
# setwd("Path_to_your_current_directory")
# source('SummarizeResults.R')
print(getwd())[1] "/Users/vinayakvsv/big_pqe/analysis/timer"setwd("/Users/vinayakvsv/big_pqe/analysis/timer")
# let's do this one at a time
cc='brca'
print(cc)[1] "brca"cur.dir = '/Users/vinayakvsv/big_pqe/analysis/timer/'
source('CancerImmunePipeline.R')[1] "setup parameters and establish the output file"
[1] "load and process gene expression data"NAs introduced by coercion[1] "removing NAs from first row"
[1] "load immune marker genes from Abbas et al., 2005"
[1] "Combine TCGA gene expression profiles with the selected reference data, remove batch effect and aggregate samples of each immune category by taking the median"Found2batches
Adjusting for0covariate(s) or covariate level(s)Standardizing Data across genesFitting L/S model and finding priors
Finding parametric adjustments
Adjusting the Data
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deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesCannot compute exact p-value with tiesthe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is 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standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe 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tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesCannot compute exact p-value with tiesError in setwd(paste(cur.dir, "scripts", sep = "/")) :
cannot change working directory# 4. Running TIMER.
# If you are interested in one cancer, please open an R console, type:
# cc='XXXX' ## replace XXXX with your desired cancer abbreviation
# cur.dir='Path_to_your_current_directory'
# source('CancerImmunePipeline.R')
#
# 5. Running TIMER in batch mode
# Open R console, type:
# setwd("Path_to_your_current_directory")
# source('SummarizeResults.R')
#print(getwd())
setwd("/Users/vinayakvsv/big_pqe/analysis/timer")
# let's do this one at a time
cc='ov'
print(cc)[1] "ov"cur.dir = '/Users/vinayakvsv/big_pqe/analysis/timer/'
source('CancerImmunePipeline.R')[1] "setup parameters and establish the output file"
[1] "load and process gene expression data"NAs introduced by coercion[1] "removing NAs from first row"
[1] "load immune marker genes from Abbas et al., 2005"
[1] "Combine TCGA gene expression profiles with the selected reference data, remove batch effect and aggregate samples of each immune category by taking the median"Found2batches
Adjusting for0covariate(s) or covariate level(s)Standardizing Data across genesFitting L/S model and finding priors
Finding parametric adjustments
Adjusting the Data
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# source('./SummarizeResults.R')# 4. Running TIMER.
# If you are interested in one cancer, please open an R console, type:
# cc='XXXX' ## replace XXXX with your desired cancer abbreviation
# cur.dir='Path_to_your_current_directory'
# source('CancerImmunePipeline.R')
#
# 5. Running TIMER in batch mode
# Open R console, type:
# setwd("Path_to_your_current_directory")
# source('SummarizeResults.R')
#print(getwd())
setwd("/Users/vinayakvsv/big_pqe/analysis/timer")
# let's do this one at a time
cc='lusc'
print(cc)[1] "lusc"cur.dir = '/Users/vinayakvsv/big_pqe/analysis/timer/'
source('CancerImmunePipeline.R')[1] "setup parameters and establish the output file"
[1] "load and process gene expression data"NAs introduced by coercion[1] "removing NAs from first row"
[1] "load immune marker genes from Abbas et al., 2005"
[1] "Combine TCGA gene expression profiles with the selected reference data, remove batch effect and aggregate samples of each immune category by taking the median"Found2batches
Adjusting for0covariate(s) or covariate level(s)Standardizing Data across genesFitting L/S model and finding priors
Finding parametric adjustments
Adjusting the Data
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standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard 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standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard 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# source('./SummarizeResults.R')# 4. Running TIMER.
# If you are interested in one cancer, please open an R console, type:
# cc='XXXX' ## replace XXXX with your desired cancer abbreviation
# cur.dir='Path_to_your_current_directory'
# source('CancerImmunePipeline.R')
#
# 5. Running TIMER in batch mode
# Open R console, type:
# setwd("Path_to_your_current_directory")
# source('SummarizeResults.R')
#print(getwd())
setwd("/Users/vinayakvsv/big_pqe/analysis/timer")
# let's do this one at a time
cc='coad'
print(cc)[1] "coad"cur.dir = '/Users/vinayakvsv/big_pqe/analysis/timer/'
source('CancerImmunePipeline.R')[1] "setup parameters and establish the output file"
[1] "load and process gene expression data"NAs introduced by coercion[1] "removing NAs from first row"
[1] "load immune marker genes from Abbas et al., 2005"
[1] "Combine TCGA gene expression profiles with the selected reference data, remove batch effect and aggregate samples of each immune category by taking the median"Found2batches
Adjusting for0covariate(s) or covariate level(s)Standardizing Data across genesFitting L/S model and finding priors
Finding parametric adjustments
Adjusting the Data
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standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard deviation is zeroCannot compute exact p-value with tiesthe standard deviation is zerothe standard deviation is zerothe standard 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deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation is zerothe standard deviation 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# source('./SummarizeResults.R')